Functional characterization of genes

ABSTRACT

Insertions into a gene of known sequence can be generated by crossing two parent plants, one of which contains a transposable element, to produce F 1  progeny plants in which the insertion is detected by means of a PCR. F 1  progeny plants containing such an insertion are self-fertilized to produce F 2  progeny which are homozygous for the insertion. The function of a gene disabled by the insertion can be ascertained from a comparison of the phenotype of the F 2  progeny with a parental phenotype. Large numbers of F 1  progeny can be tested simultaneously for the presence of insertions. A collection of F 2  seed can be stored and used for phenotype comparison when an insertion is detected.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/835,638 filedApr. 10, 1997 now U.S. Pat. No. 5,962,764 which is a continuation ofU.S. Ser. No. 08/262,056 filed Jun. 17, 1994, (now abandoned).

BACKGROUND OF THE INVENTION

Modern advances in recombinant DNA technology have made possible thecloning and sequence determination of many genes, the functions of whichremain to be determined. Methods have been developed for ascertainingthe functions of these genes and may be divided into three main types.

The first method involves the sequestration of the messenger RNAcorresponding to the gene sequence of interest by an antisenseoligonucleotide or nucleotide analogue complementary to the sequence ofthe RNA. This prevents initiation of translation of the messenger RNA bythe ribosome, and production of the protein product of the gene isthereby suppressed. The function of the gene is therefore imputed fromobservation of a mutant phenotype induced by this suppression.Introduction of the antisense oligonucleotide may be achieved either bytransfection of the target cell with a suitable gene construct whichdirects production of an antisense transcript within the cell, or bydirect introduction of exogenous oligonucleotides or oligonucleotideanalogues into the cell.

Although the antisense method has been successful in model systems suchas cultures of dissociated cells, its implementation in a whole organismis problematic. For example, simultaneous delivery of exogenousoligonucleotides to all tissues of interest in a whole organism isextremely difficult. Exogenous oligonucleotides are also metabolized bycells, and any phenotype produce by this method is therefore onlytemporary. To preserve a desired mutant phenotype for prolonged studythe mutation must be made heritable by creation of a transgenicorganism. This requires that a gene construct coding for the antisenseRNA be stably creation of a transgenic organism. This requires that agene construct coding for the antisense RNA be stably transfected intothe germ cells of the organism under study, followed by rounds ofinbreeding of transgenic progeny to make organisms homozygous for themutation. Creation of transgenic organisms is therefore typically alaborious and expensive process.

A second approach to determine the function of a gene of known sequenceis by the “co-suppression” technique, which also requires the creationof a transgenic organism. In this approach the transfected geneconstruct codes for the gene of interest in the sense orientation, and asmall proportion of the transformants exhibit loss of function of thegene of interest via an unknown trans mechanism. As with the antisenseapproach this method suffers from the disadvantage of requiring thecreation of a transgenic organism.

A third approach is to disrupt the gene of interest by transformation oftarget cells with a vector designed to stably integrate into the hostchromosome within the coding sequence of the gene of interest, via theprocess of homologous recombination. Since homologous recombination is arare event, this approach typically utilizes a targeting vector designedto introduce a gene for antibiotic resistance normally lacking in thetarget cells, allowing selection of the small number of desiredtransformants. Integration of the antibiotic resistance gene within thecoding sequence of the gene of interest in this fashion also serves todisrupt normal transcription of the gene, producing an aberrant,non-functional protein product. This approach also requires germ-linetransmission of the disrupted gene for subsequent generation oforganisms homozygous for the mutation, and therefore also suffers fromthe disadvantages discussed supra.

A more recent method to cause gene disruption has been the use ofnaturally occurring transposable elements to introduce gene insertions,together with the use of a PCR to detect an insertion event within aparticular gene of interest. Ballinger et al., Proc. Nat'l Acad. Sci.86: 9402-06 (1989); Kaiser et al., loc. cit. 87: 1686-90 (1990); Zwaalet al., loc. cit. 90: 7431-35 (1993). In this method two oligonucleotideprimers were employed in the PCR, with one primer complementary to asequence within a particular gene of interest, and the other primercomplementary to a portion of the tandem inverted repeat sequence of thetransposable element.

Both Ballinger et al. and Kaiser et al. crossed parental strains of thefruit fly Drosophila melanogaster, one strain of which carried atransposable element, to produce a pool of heterozygous F₁ progenybearing genome insertions caused by the transposable element. Ballingeret al. then screened the genomic DNA of the F₁ progeny for the presenceof insertions in two genes of unknown function, as described above.Flies in which the desired gene insertions were observed were then usedto produce F₂ progeny homozygous for the insertion, and their phenotypeexamined for effects attributable to the absence of function of the geneof interest. But no phenotypic changes from the wild type were observedfor any F₂ flies which were homozygous for insertions into either geneof interest.

Kaiser et al. carried out the screening for gene insertions using theDNA of the segregating F₂ generation of flies, rather than at the F₁generation. Insertions were observed into a gene which was previouslyknown to produce a specific phenotype when disrupted by insertion, andthe expected phenotype was observed. In this fashion Kaiser et al.demonstrated that disruption of a known gene by insertion of atransposable element could be correlated with observation of apreviously known phenotype.

An additional problem associated with DNA analysis of the F₂ generationinstead of the F₁ generation is the possibility of observing additionalinsertion events into the gene of interest caused by the activity of thetransposable element during generation of the F₂ generation. Thesemutant alleles will be detected by DNA analysis of the F₂ generation,but as they will not genetically segregate until the next gametic cyclethey will not contribute to the homozygous genotype of the F₂generation. Accordingly, DNA sampling at the F₂ generation will lead tothe identification by PCR of insertion events in the gene of interestwhich will not be reflected in the phenotype of the F₂ generation,generating false positive results which require further experimentationto detect.

Zwaal et al., working in the worm Caenorhabditis elegans (C. elegans),also carried out PCR analysis for gene insertion events caused by atransposable element in the DNA of the F₂ and subsequent generations. Inaddition the Tc1 transposable element was used which also generateddeletion mutants in the F₂ and subsequent generations caused by thetransposable element “jumping” from the initial site of insertion andexcising some amount of DNA from the region of the gene flanking theinsertion site. Despite the detection of 23 insertion events into 16known genes and 7 deletion events in 6 known genes, no phenotypicdifference from the wild type was observed in any of the F₂ worms orsubsequent generations.

Zwaal et al. also described the production of libraries of F₂ progeny ofC. elegans in a frozen state. These libraries could be used both toprepare DNA for PCR analysis as described above, and to recover viableworms for subsequent phenotypic analysis. Use of such libraries could inprinciple obviate the need to generate new collections of mutant progenyin order to analyze the function of each new gene of interest. Thelibraries disclosed, however, allow the preparation of only smallquantities of DNA which may be sampled only a limited number of times,and which are insufficient for distribution to other laboratories. Inorder to detect insertions in more than a small number of genes,therefore, frequent generation of new libraries will still be required.

Although the aforementioned techniques are available for determining thefunction of a gene of known sequence, each conventional methodology hassignificant drawbacks, and the development of a rapid, inexpensivemethod would be highly desirable.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodfor ascertaining the function of a gene for which the sequence is known.

It is also an object of the present invention to provide products,including genomic DNA collections and seed assemblages of particularconstituency, that are particularly adapted to implementing such amethod.

In accomplishing these objects, there has been provided, in accordancewith one aspect of the present invention, a collection of genomic DNAsprepared from individual F₁ progeny plants obtained from crossing twoparent plants, one of which contains a transposable element sequence,such that at least some of the F₁ progeny plants are heterozygous forinsertion of the transposable element at a gene of known sequence, andwhere genomic DNA from each F₁ plant is present separately in thecollection. In one preferred embodiment the collection is containedwithin the wells of a microtiter plate configured to allow automatedsampling of the genomic DNA for PCR.

In accordance with another aspect of the present invention, there hasbeen provided a collection of F₂ seed obtained by self-fertilization ofthe aforementioned F₁ progeny plants.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a novel method for determining thefunction of a gene of known sequence, and also provides products thatfacilitate the practice of the invention.

The invention is based on generating insertions into a gene of knownsequence by crossing two parent plants, one of which contains atransposable element, to produce F₁ progeny plants in which theinsertion is detected by means of a PCR. F₁ progeny plants containingsuch an insertion are self-fertilized to produce F₂ progeny at leastsome of which are homozygous for the insertion. The function of a genedisabled by the insertion can be ascertained from a comparison of thephenotype of the F₂ progeny with a parental phenotype. Large numbers ofF₁ progeny may be tested simultaneously for the presence of insertions.The F₂ seed can also be stored and used for phenotype comparison when aninsertion is detected in the genome of the corresponding F₁ plant. The1:1 correspondence of DNA from an individual plant and the progeny fromthat individual plant is unique to this method.

In accordance with the present invention, a collection of genomic DNAsare prepared from numbers of F₁ plants produced by the crossing of twoparent plants, one of which parents contains a transposable element.Sufficient DNA is prepared from each F₁ plant that the DNA samples canbe used repeatedly to analyze for the presence of insertions into alarge number of genes of interest, without the need to repeatedlygenerate new generations of F₁ or progeny plants. The invention relieson the use of a highly active transposable element which causes manyinsertion events per gametic cycle, thereby reducing the number of F₁plants which must be produced in order to ensure an insertion occursinto any given gene of interest. In particular the inventioncontemplates the use of the highly active Mutator (Mu) family oftransposable elements as a means of minimizing the number of F₁ plantsrequired to ensure a desired insertion event. If a less activetransposable element is used, correspondingly greater numbers of F₁plants must be produced to produce the desired insertion.

Individual F₁ progeny plants are self-fertilized to produce a collectionof F₂ seed, some of which F₂ plants will be homozygous for the geneinsertion event of interest. The F₂ seed from each F₁ plant is keptseparate, and these seed may be stored for extended periods of time forlater germination and subsequent phenotypic analysis whenever a geneinsertion event is detected in the corresponding F₁ parent plant asdescribed above.

The present invention also provides a simple means of generating geneinsertions which are heritable. Mutant phenotypes produced by the methodof the invention may thus be preserved for further study by maintainingplants homozygous for the gene insertion. In addition, mutations thatdisrupt gametophyte development or function, or that are lethal in thehomozygous condition, can be recovered and maintained in theheterozygous condition.

The present invention thus provides a rapid and inexpensive method ofgenerating and detecting mutations in a gene of known sequence, whichmutations cause loss of function of the gene. In particular, the presentinvention allows a function to be assigned to the gene by correlationwith an observed mutant phenotype in organisms homozygous for themutation.

A. Generation of F₁ Plants Heterozygous for Gene Insertion Events Causedby a Transposable Element

An initial step in a method according to the present invention involvesthe crossing of two parent plants, one of which carries a transposableelement, to produce a collection of F₁ progeny bearing gene insertionscaused by the activity of the transposable element. A transposableelement is a member of a class of diverse DNA segments that can insertinto nonhomologous DNA in a manner independent of the generalrecombination function of the host. Most transposable elements carryterminal inverted repeat (TIR) sequences. These are DNA segments locatedat each terminus of the transposable element which share an identicalsequence but are inverted with respect to each other. The presentinvention contemplates, but does not require, the use of a transposableelement carrying a TIR.

Via the aforementioned cross, F₁ progeny are obtained by standardmethods. In a preferred embodiment the plants are maize plants, and oneparent plant carries the Mu family of transposable elements. Mu is themost mutagenic transposable element known. The other parent plant ispreferably an inbred or hybrid strain, and in preferred embodiments isthe B73 or A632 inbred strain or the Pioneer 3394 hybrid strain ofmaize. Insertion events caused by the transposable element occur atdispersed loci within the genome of the F₁ progeny plants produced bythe crossing of the parent plants.

In a preferred embodiment, sufficient F₁ progeny are produced to ensurea high probability that many insertion events will occur in any givengene. For example in maize containing Mu, it is estimated thatproduction of 10⁵ F₁ progeny will generate enough independent mutationsto ensure a high probability that many insertion events will occur inany given gene.

B. Isolation of genomic DNA from the F₁ progeny

Genomic DNA can be isolated from plants using techniques that are wellknown in the art. See, for example, Taylor et al., “Isolation andcharacterization of Plant DNAs” in METHODS IN PLANT MOLECULAR BIOLOGYAND BIOTECHNOLOGY, Glick et al. (eds.), pages 38-41. For example, planttissue can be ground after freezing in liquid nitrogen, extracted with abuffer containing the detergent CTAB, clarified by centrifugation toremove insoluble debris, and the resultant solution extracted withchloroform and isoamyl alcohol. DNA is precipitated by addition ofbuffer containing CTAB, collected by centrifugation, resuspended andpurified on a cesium chloride density gradient. See Taylor et al.,supra, at pages 38-41.

In a particularly preferred embodiment, six lyophilized leaf punchescollected in 1.2 milliliter polypropylene tubes would be positioned in96-well plates, with the positions recorded for identification of thesource of the leaf punch. In a preferred method of DNA extraction, twostainless steel oil-free ball bearings are added to each tube containingthe 6 leaf punches. The tubes are centrifuged briefly to position theballbearings at the bottom of the tube, and the tubes are then sealedwith mylar using a heat sealer. Using an apparatus capable of generatingthe necessary shaking movement, such as a modified jigsaw, the 96-wellplates are shaken so that the integrity of the tissue is destroyed bythe ball bearings. Six hundred microliters of extraction buffer areadded to each tube, and the tubes are resealed and shaken again.

The plates are then heated for approximately 45 seconds in acommercially available microwave oven at high power, and then spun in acentrifuge at approximately 4,000 rpm for approximately 15 minutes. Thesupernatant is then added to fresh tubes containing isopropanol. Thetubes are then mixed and centrifuged. The isopropanol is then decantedand as much isopropanol as possible is removed. The resulting pelletsare dried, and 50 microliters of storage buffer may then be added, atwhich time the tubes are heated at 65° C. for 10 minutes to resuspendthe pellet. For storage, the plate may be sealed with mylar using a heatsealer at approximately 380° C. and stored at −80° C.

In light of the fact that the present invention is directed to geneticanalysis of large numbers of samples, at this point the originalcollection plates will have gone through a liquid handler and beenreconfigured into either 4× or 9× plates. These are plates in which eachwell in the conventional 96-well microtiter plate format is replacedwith a square array of 4 or 9 smaller wells, comprising respectively 384or 864 wells in total. Using computer assisted tracking devices, anysingle well in the 9× plate can be referenced back to a well on theoriginal 96-well plate from which a particular sample was obtained. Inpreferred embodiments of the invention, a 9× microtiter plate willtypically be used, but the present invention also envisions use of 4×plates.

C. Analysis of the Genomic DNA from the F1 Progeny Plants by PCR

Genomic DNA isolated from the F₁ plants is used as target DNA for a PCR,where insertion of a transposable element within a gene of knownsequence is detected by using one primer complementary to the gene ofinterest, and, in a preferred embodiment, one primer complementary tothe terminal inverted repeat (TIR) sequence of the transposable element.Because the sequences of the terminal repeats of the transposableelement are identical but inverted in orientation with respect to eachother, a single PCR primer complementary to the repeat sequence willprime in both directions from the transposable element, ensuring thatgeometric amplification of DNA will occur, irrespective of theorientation of the transposable element, provided that the gene-specificprimer anneals to the target DNA molecule within a sufficient distanceof the annealing site of the TIR-specific primer. Primers complementaryto non-palindromic insertion sequences of the transposable element mayalso be used. In this case, since the insertion may occur in eitherorientation, primers complementary to both strands of the transposableelement are required to ensure detection of insertion events. Suitableoligonucleotide primer sequences may be designed by methods known in theart, based on the known sequences of the gene of interest and the TIR ofthe transposable element. See for example, Rychlik “Selection of Primersfor Polymerase Chain Reaction” in METHODS IN MOLECULAR BIOLOGY, VOL. 15:PCR PROTOCOLS: CURRENT METHODS AND APPLICATIONS, B. A. White (ed), pages31-40.

The PCR reaction can be run using protocols well known in the art. Seefor example, Delidow et al. “Polymerase Chain Reaction: Basic Protocols”in METHODS IN MOLECULAR BIOLOGY, VOL. 15: PCR PROTOCOLS: CURRENT METHODSAND APPLICATIONS, B. A. White (ed), pages 1-29. In a preferredembodiment of the present invention, an appropriate volume of PCRreaction solution is added to each well of a 9× microtiter plate. Thismay be done either manually or using a robotic liquid handler. Next, adefined amount of genomic DNA is added to the PCR reaction solution ineach well, using either a pinner tool or manual addition techniques.

In preferred embodiments, unique steps are taken prior to addition ofPCR reaction solution and DNA to the wells of the microtiter plates inorder to prevent formation of gas bubbles during PCR. Due to the smallvolumes of the solutions used in the PCR the presence of bubbles cancause loss of sample during thermocycling. Hence, particular unique PCRbuffers are used as described below, the microtiter plates are preheatedprior to use, degassed water is used, and the plates are thencentrifuged briefly prior to placing in an oven for the PCR reaction.

In a particular preferred embodiment, samples of DNA from 9× plates,each well of which contains genomic DNA from a single plant, arecombined into pools containing DNAs from 48 individual plants. Thesepools are then transferred to a 9× plate, so that each well of the 9×plate contains DNAs from 48 plants, and in such a way that the contentsof each pool may be referenced back to a collection of individual plantDNA samples. A 9× plate prepared in this manner is thus capable ofholding DNAs from 41,472 plants. In a preferred embodiment, one third ofthe wells on the 9× plate are used for control samples, and each platetherefore can hold DNAs from up to 27,648 plants.

In an alternative preferred embodiment, 12 leaf punches are taken fromeach plant. Six punches from each plant are used to prepare individualDNA samples as described above. The remaining 6 leaf punches arecombined in pools of punches from 48 different plants, and a mixed DNAsolution is prepared from each pool as described above, except that theDNA is prepared in a 15 ml tube, using 6 ml of extraction buffer. Themixed DNA solutions are used to prepare 9× plates identical to thoseprepared by mixing the DNA samples as described previously.

Detection of PCR products may be carried out using methods well known inthe art. See, for example, Allen et al., “The use of the PolymeraseChain Reaction and the detection of Amplified Products,” in METHODS INMOLECULAR BIOLOGY, VOL. 15: PCR PROTOCOLS: CURRENT METHODS ANDAPPLICATIONS, B. A. White (ed), pages 113-128. In a preferred embodimentPCR amplification products are detected by transferring samples ofamplified DNA to a blotting membrane made of a suitable material such asnylon, followed by hybridization with a radioactively labeled probederived from the gene of interest.

In a particularly preferred embodiment, following PCR the contents ofthe microtiter plates are transferred to a nylon membrane. Twomembranes, which are preferably 8×12 cm, are presoaked in sterile,glass-distilled et al., “The use of the Polymerase Chain Reaction andthe detection of Amplified Products,” in METHODS IN MOLECULAR BIOLOGY,VOL. 15: PCR PROTOCOLS: CURRENT METHODS AND APPLICATIONS, B. A. White(ed), pages 113-128. In a preferred embodiment PCR amplificationproducts are detected by transferring samples of amplified DNA to ablotting membrane made of a suitable material such as nylon, followed byhybridization with a radioactively labeled probe derived from the geneof interest.

In a particularly preferred embodiment, following PCR the contents ofthe microtiter plates are transferred to a nylon membrane. Twomembranes, which are preferably 8×12 cm, are presoaked in sterile,glass-distilled water. The membranes are placed one at a time onto thetop of the microtiter plate. Placement should be done with care so thatno air bubbles remain trapped between the plate and the membrane, orbetween membranes. Blotting pads are then placed an top of the membranelayers, followed by a sheet of plexiglass, and the assembly is clampedand placed upside-down into a centrifuge. The blotting assemblies thenare centrifuged to transfer the PCR-amplified DNA to the membranes, andthe membranes carefully separated from one another. Prior to removingthe last membrane the plate is placed back in the centrifuge right sideup and centrifuged briefly again in order to remove the oil from themembrane. The DNA on the resulting membrane or “dot blot” is thencross-linked to the membrane, denatured and the membrane is prepared forhybridization.

The next step in the method of the present invention is hybridizationwith an appropriate labeled nucleic acid probe. Suitably labeled probesmay be prepared and hybridized to the membranes containing the PCRproducts by methods known in the art. See, for example, CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. (eds), pages 2.10.2-3 and3.5.9-10. The membrane is then exposed to film (if a radioactivelylabeled oligonucleotide is used) in order to detect hybridization.Appropriate scanning software may be utilized in order to scan theresulting autoradiographs.

When hybridization of the labeled probe to one of the pools of DNAdescribed above is observed, the PCR is repeated using as template thesamples corresponding to the individual DNAs making up the pool.Transfer of the amplified products to a membrane and hybridization isthen repeated as above to determine which individual DNA sample wasresponsible for the positive hybridization signal. Individualamplification products which give positive hybridization signals arethen size-fractionated by agarose gel electrophoresis, with detection bystaining with ethidium bromide. Observation of a discrete PCR product inthe gel indicates that an insertion event has occurred within, or closeto, the gene of interest. Confirmation that an insertion event occurredwithin a gene or a specific segment of a gene, rather than outside, canbe obtained by repeating the PCR using additional gene-specific primerstogether with primers complementary to the transposable element. Sincethe structure of the gene of interest is known, analysis of the size ofthe products thus obtained and comparison with the sizes expected fromthe gene structure allows the site of the insertion to be estimated.Insertions that disrupt gene function can be selected by using primersthat will identify insertions in the promoter region of the gene or inexons.

D. Production of F₂ progeny and assessment of phenotype

The F₁ progeny plants described above may be self-fertilized by standardmethods known in the art to produce F₂ progeny. This step may be carriedout prior to or subsequent to the DNA analysis described above. An F₁plant which is heterozygous for gene insertions produced by atransposable element will, when self-fertilized, produce viable andnon-viable F₂ progeny at least some of which will be homozygous for thisgene insertion. When an insertion is detected in a gene of interest in aplant of the F₁ generation, the F₂ progeny of that plant are analyzedfor a phenotype which differs from the expected wild-type phenotype, andwhich may be attributed to inactivation of the gene of interest by theinsertion. A collection of F₂ seed can be prepared and stored forsubsequent phenotypic analysis whenever mutations are identified in thecorresponding collection of F₁ DNAs. The capability of generating largeamounts of DNA from individual F₁ plants, taken together with the largenumbers of F₂ seed produced from each F₁ plant means that analyses forinsertions in a great many genes may be carried out without the needeither to generate new F₁ progeny or to do breeding with the siblings orsubsequent generations.

Phenotypic analysis of the F₂ plants may be carried by methods which arewell known in the art. For example, visual examination and physicalmeasurements will detect any gross changes in size or shape of any partof the plant. Similarly, microscopic examination will reveal changes instructural features which cannot be seen with the naked eye.

Phenotypic changes at the molecular level may be revealed by analysis ofthe expression of the mRNA or the protein product corresponding to thegene of interest. For example, patterns of expression of RNA in planttissue may be examined by means of in situ hybridization, using a DNA orRNA probe specific for the gene of interest. Levels of expression of themRNA corresponding to the gene of interest may be measured by means ofNorthern blots, using mRNA or total RNA preparations prepared from thewhole plant or from particular organelles of the plant. Similarly mRNAcorresponding to the gene of interest may be detected by the reversetranscription PCR method. This method involves reverse transcription ofthe mRNA, followed by second strand synthesis and PCR amplification ofthe resulting double-stranded DNA. See, for example, Shuldiner et al.“RNA Template-Specific Polymerase Chain Reaction” in METHODS INMOLECULAR BIOLOGY, VOL. 15: PCR PROTOCOLS: CURRENT METHODS ANDAPPLICATIONS, B. A. White (ed), pages 169-176, and references therein.

Analysis of patterns of expression of the protein product of the gene ofinterest in plant tissue may be achieved by means ofimmunohistochemistry, using a monoclonal or polyclonal antibodypreparation specific for the protein of interest. Levels of expressionof the protein corresponding to the gene of interest may be measured bymeans of Western blots, using protein prepared from the whole plant orfrom particular organelles of the plant. Those of skill in the art canreadily devise other methods of analyzing the phenotype of the F₂ plantsdescribed above. It should also be noted that the present inventionpermits all members of a gene family to be disrupted. Although anysingle disrupted gene may not produce a mutant phenotype, combinationsof double mutants can be easily made, using conventional breedingtechniques, and detected by PCR as described above.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1

Preparation of Genomic DNA from F₁ Plants

Zea mays leaf punches were collected using a handheld leaf punch andlyophilized. Six punches of leaf tissue were collected per plant, in 1.2ml polypropylene tubes, and placed in appropriate positions in 96-wellplates. Two stainless steel, oil-free ballbearings ({fraction (5/32)} ofan inch) were added to each tube. The tubes were centrifuged briefly toposition the ballbearings at the bottom of the tube.

Using a flat surface, all of the tubes in the 96-well plate were firmlypressed down so that the tops of the tubes were level. The tubes werethen sealed with mylar using a heat sealer at approximately 380° C. Theplates were then shaken for 30 seconds using a modified jigsaw adaptedfor rapid, high torque shaking of the sample tubes. The plates wereshaken for 30 seconds to macerate the plant tissue, and then 600 μl ofextraction buffer was added to each tube. The extraction bufferconsisted of 0.2 M trisodium citrate, 0.01 M DTPA(diethylenetriaminepentaacetic acid) (free acid), 0.8 M LiCl, 0.5% PEG(polyethylene glycol 8000), and 0.005M o-phenanthroline monohydrate. DNAextraction buffer was sterile filtered before use, and stored in darkplastic without a stir bar at 4° C. The tubes were resealed, shakenagain as above, and then heated for 45 seconds per plate in aconventional microwave oven at high power. The plates were thencentrifuged for 15 minutes at 4000 rpm. A storage plate was thenprepared containing 120 μl isopropanol. 200 μl of the supernatant fromthe spun sample tubes was then added to 120 μl of the isopropanol. Thetubes containing the isopropanol/supernatant mixture were mixed and spunin a centrifuge at 4000 rpm for 15 minutes. Following centrifugation,the isopropanol was decanted, and the plates tipped upside-down andtapped on blotting paper in order to remove as much isopropanol aspossible. The pellets were dried for approximately two hours. When thepellets were dry, 50 microliters of storage buffer were added to eachwell. The storage buffer consisted of 10 mM Tris base, 10 mM EDTA(disodium), 0.01% Triton-X 100, with a final pH of 8.0 adjusted withNaOH. The Triton-X 100 may be left out if the DNAs will not betransferred using a pinning apparatus. Following addition of storagebuffer, the microtiter plates were heated at 65° C. for 10 minutes toresuspend the pallets. Following heating, the microtiter plates weresealed with mylar using a heat sealer at approximately 380° C.

EXAMPLE 2

Amplification by PCR of F₁ DNA

Polymerase chain reaction was performed in 9× microtiter plates. Theplates were stored at 65° C. to remove dissolved gases from the plates.Once plates have cooled to room temperature sufficient gas can reabsorbwithin a day to cause bubble formation in the PCR reaction.Alternatively, the plates may be degassed, filled, and then stored in anair tight manner so as to avoid reabsorption. In addition, a PCR bufferwas used containing 0.01% Triton-X 100, and water which was degassed byboiling and storage under vacuum. In addition, immediately prior toplacement of microtiter plates in the oven for PCR the microtiter plateswere spun at approximately 2000 rpms for 30 seconds to remove bubbles.Alternatively, it is possible to visually monitor the plates for bubbleformation at the bottom of the wells at approximately 60 minutes intothe PCR reaction run. If bubbles are present, they can be removed by abrief spin in the centrifuge. The reaction cycle is interrupted as itbegins the cooling phase, the plates are spun, and immediately returnedto the oven.

The PCR “Hot Tub” buffer used in these PCR reactions was designedspecifically to overcome the challenges of high heat, changes ofmagnesium (Mg) ion concentration, and low amounts of DNA template. Useof the buffer described in these experiments has significant advantagesover the buffer supplied with the commercial “Hot Tub” enzyme when PCRreactions are run in Biotherm™ ovens, with less DNA than optimal, orwith some carryover chelator from the template DNA preparation.

Citrate has a K_(m) for Mg ions at about the desired concentrationrequired for the Hot Tub enzyme. It thus acts as an excellent Mgconcentration buffer. About half of the 20 mM Mg in the buffer ischelated by the citrate, leaving the final Mg concentration stabilizedat about 10 mM. Sucrose was determined to be the most effective reagentto stabilize the polymerase enzyme at high heat. When carrying out a PCRin Biotherm ovens, some of the outer wells may overheat when bringingthe interior wells to the required temperature. Addition of sucrose tothe buffer protects the polymerase enzyme in these outer wells.Furthermore, MgSO₄ is used instead of MgCl, as the presence of chlorideion has shown deleterious effects on the outcome of the PCR. Tris baseand glycine are used to buffer the pH of the PCR solution at pH 9.2.

Histidine and other secondary and higher amines act as oxygen radicalquenchers, and were shown to reduce the requirement for template DNAconcentrations to well below 1 ng/μl of PCR reaction mixture. Finally,Triton-X 100 appears to have some enzyme heat stabilizing affect, and asnoted above contributes greatly to elimination of bubbles. In its finalform the Hot Tub PCR buffer consists of the following:

Tripotassium Citrate 20 mm MgSO₄ 20 mM Tris base 40 mM Glycine l0 mML-Histidine  5 mM Triton X-100 0.01%

The pH of the buffer is adjusted to 9.2 with NaOH or KOH. Heating of thebuffer is required to dissolve the histidine and sucrose. The buffershould not be filtered and should be stored at −20° C.

Four microliters of PCR reaction solution were added to each well. Forlarge scale preparations a liquid handler, for example an IVEKTM machinecan be used. The IVEK™ pumps were set at a setting of 7 in order todeliver approximately 4 μl/stroke. Only DC-200 silicone oil (Fluka)should be used. The oil may be made blue using approximately 200 mg ofSolvent Blue 35 (Aldrich). When using IVEK™ pumps, bubbles in the tubingare to be avoided. The IVEK™ was debubbled using distilled water, thenloaded with 1× PCR buffer. The PCR mix was then prepared and placed inthe tubing. The tube was then placed on the input port of the IVEK™without introducing bubbles into the tube. The PCR mix used in the IVEK™machine for a PCR reaction was as follows:

primer I 1 μM primer 2 1 μM dNTP 240 μM 10X Hot Tub buffer 1X SchillingYellow 1% Hot Tub enzyme 0.15 U/4 μl Degassed sucrose 10%

The enzyme should be added last to a premixed solution. The dNTPs werefound to be unstable, and were kept in single use containers at −80° C.and thawed only once. Primers are made to 100 μM as they are received,and stored at −20° C.

One nanogram of genomic DNA template per microliter of the PCR reactionsolution was added to each well, using either a pinner tool or manualaddition methods. In the present experiments, a specifically designedpinner tool was used to move small amounts of many samples with a highdegree of reliability. Standard pinners have minimal surface area andwill not hold small drops of liquid reliably. The pins used in theseexperiments were stainless steel Brasseler dental burrs (friction gripH1-010 US #2) with carbide tips. Each tip has 6 concave slots which holdabout 8 nl of liquid for a total of about 50 nl per pin. The pin willdisplace approximately 8 μl of the 20 μl volume capacity of a 9× platewell. Consequently, the amount of DNA solution in the well can be up to12 μl. Wells containing 12 μl of DNA solution can thus be accessed over200 times. A recommended DNA concentration for the solution is 100ng/μl. Taken together with the large amount of DNA available from theleaf punches, as described in Example 1, this means that each pooled DNAsolution in the 9× plate may be sampled for PCR at least 10,000 times.The slots at the tip of the burr are too small to accept liquids withhigh surface tension, such as water, and hence a surfactant must be usedto reduce the surface tension or the pinner will function unreliably.Triton-X 100 at 0.01% was determined to be optimal, and therefore wasincluded in the DNA storage buffer. The pins were designed so that allof the liquid is stored at the tip. Because of this, the volume held bythe tip does not decrease as the volume of the DNA solution decreases,and the pins are extremely effective at moving the last quantities ofthe DNA solution from the well. Alternative pins for use in the methodof the invention are the 6801-010 diamond burr pins. The many internalangles between the diamond and the embedding epoxy of such pins givesthis tip more reliability than the six internal slots of the carbideburr.

In order to carry out the pinning, the pinner was fastened securely to ajig. The DNA source plate and the reception plate containing the PCR mixwere placed in the jig in the same orientation, so that appropriatewells were pinned with the matching pin. The pinner was pressed into theDNA plate two or three times in order to wet the pins. The pinner wasthen moved to the reception plate and pressed into the plate three orfour times fairly vigorously in order to move the DNA solution throughthe oil overlay. The pinner must not be returned to the DNA source plateafter it is contaminated with oil and PCR mix, and must be thoroughlycleaned before reuse. The PCR plate was then spun at 2000 rpm for a fewseconds in order to place the PCR mix and oil at the bottom of eachwell. The PCR was carried out in a Biotherm™ oven. The followingsuggested profile presents temperatures as measured by the oven'stemperature probe in the air inside the oven:

Time Temp Ramp Speed 2 min 99 2° C./sec 1 min 96 2° C./sec 3 min 55 2°C./sec 6 min 72 2° C./sec

The initial 99° setting was to move the plate toward a denaturationtemperature. As the edges of the plate approach 96° C. the temperaturewas dropped to 96° C. for one minutes in order to allow the center ofthe plate to catch up with the edges. The same concept was applied incooling the plate. As the edges of the plate approach approximately 55°C., the air temperature was brought to 55° C. to equilibrate the centerof the plate, and the temperature was then moved slowly through thelikely oligonucleotide hybridization temperatures. Unusually long timesat the extension temperature may sometimes be advantageous. The Hot Tubenzyme has a temperature optimum at 70° C., and this temperature wasobtained with an approximate oven air temperature of 72° C.

EXAMPLE 3

Detection of Amplified DNA Products

Due to the fact that the signal and signal/noise ratio limiting aspectof the method of the present invention is the amount of PCR DNA on themembranes, and further due to the fact that this amount of DNA is alsolimiting for hybridization time, spin or centrifugation “dot blotting”was developed as an alternative to pinning dot blots. This alternativewas found to be superior because it can transfer all of the PCR solutionto the filter much faster than the repeated applications which arerequired by using a pinner. This method also overcomes the problems ofdiscarding the oil overlay.

Following PCR the microtiter plates were removed from the Biotherm™ovens. A series of membrane and blotting pad layers were then overlaidon the top of the PCR microtiter plates. This was accomplished in thefollowing manner: first, the membranes were labeled for identificationand orientation using a VWR biotech marker on a uniform selected side.Zeta-Probe (Bio-Rad) membranes were rinsed and soaked in distilledwater. The PCR microtiter plate was wiped with a tissue in order toremove excess surface oil. Two membranes were then blotted to removestanding water and placed on the top of the PCR plate in the appropriateorientation, and covered with a plastic sheet. The plastic surface isthen rubbed to remove excess water and any entrapped air bubbles betweenthe filters or between the filters and the plate. A wetted blotting padwas blotted to remove standing water, placed on top of the plasticcovered membrane layers, and then itself covered with plastic and rubbedin order to force contact with the membranes. A second blotting pad,this one dry, was placed an top of the water soaked blotting pad, andthe entire plate-membrane-blotting pad structure was inverted andcentrifuged at approximately 1800 rpm for two minutes, or until littleor no liquid phase remains in the plate. The sandwich was then removedfrom the centrifuge, and the two blotting pads were removed anddiscarded. It was found to be critical at this stage not to disturb themembranes on the plate. The oil which had been forced against themembrane was replaced into the plate by reinverting the sandwich andcentrifuging the oil to the bottom of the wells at 1800 rpm for a fewseconds.

The membranes then were removed and pre-treated for hybridization. Themembranes were placed DNA-face up carefully on the surface of a blottingpad soaked with 0.6 M NaCl, 0.4 M NaOH for about two minutes. Filterswere then moved again, DNA face up, to a pad soaked with 0.5 M Tris atpH 7.5, and 1.5 m NaCl. Air bubbles between the membrane and theblotting pad were detected by watching the color of the red dye on themembrane. This dye is a pH indicator. The membranes were then fixed tothe membranes using a Stratalinker (Stratagene) at 200 μJ, and baked ina vacuum oven at 80° C. for 2 hours.

Although Zeta-Probe filters were found to work particularly well in themethod of the invention, other positively charged nylon filters wouldalso be suitable. The blotting pads must be soft and smooth enough toconform tightly to the surface of the membrane, and porous enough toallow liquid to easily pass through.

Preparation of radiolabeled probe by the standard random hexamer primingprotocol method and hybridization to the membranes were both carried outby standard methods known in the art. See, for example, CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. (eds), pages 2.10.2-3 and3.5.9-10.

What is claimed is:
 1. A two-component product for the functionalcharacterization of a gene of known sequence, comprising: (a) a firstproduct, said first product being a collection of F1 progeny genomic DNAheterozygous for a plurality of insertion sequences within or close to aplurality of different genes, said first product made by the process ofcrossing two parent plants, at least one of said parent plantscomprising within its genome at least one insertion sequence, whereinsaid crossing yields a plurality of F1 progeny collectively containing aplurality of said insertion sequences within or close to a plurality ofdifferent genes such that at least one of said F1 progeny isheterozygous for said insertion sequence into or near said gene of knownsequence; and, (b) a second product, said second product being acollection of F2 progeny made by the process of self-fertilizing saidplurality of F1 progeny, wherein the product of (a) and (b) arefunctionally related such that each F1 progeny genomic DNA of (a) isindexed to its F2 progeny of (b).
 2. The two-component product of claim1, wherein said plants are corn plants.
 3. The two-component product ofclaim 1, wherein said insertion sequence is a transposable element. 4.The two-component product of claim 1, wherein said insertion sequence isfrom the Mutator family of transposable elements.
 5. The two-componentproduct of claim 1, wherein said insertion sequence comprises terminalinverted repeat sequences.
 6. The two-component product of claim 1,wherein said F1 progeny genomic DNA is present separately in saidcollection.
 7. A method for the functional characterization of a gene ofknown sequence, comprising: (a) providing a collection of genomic DNAfrom a plurality of F1 plants at least some of which are heterozygousfor an insertion sequence within or close to said gene, wherein saidplurality of F1 plants are produced by the process of crossing twoparent plants at least one of which contains within its genome aninsertion sequence; (b) providing a plurality of F2 progeny produced bythe process of self-fertilizing said plurality of F1 plants, whereingenomic DNA from each of said F1 plants is indexed to its F2 progeny;(c) determining from said collection of (a) said F1 genomic DNAcontaining said insertion sequence within or close to said gene by PCRamplification using two oligonucleotide primers, with one of saidprimers annealing to said gene and the other primer annealing to saidinsertion sequence, and whereby said PCR amplification is obtained onlywhen said insertion sequence occurs within or close to said gene; and,(d) indexing said F2 progeny of (b) from said F1 genomic DNA containingsaid insertion sequence in said gene as determined in (c), wherein amutant phenotype of said indexed F2 progeny of (d) is associated withthe function of said gene of known sequence.
 8. The method of claim 7,wherein said plants are corn plants.
 9. The method of claim 7, whereinsaid insertion sequence is a transposable element.
 10. The method ofclaim 7, wherein said insertion sequence is from the Mutator family oftransposable elements.
 11. The method of claim 7, wherein said insertionsequence comprises terminal inverted repeat sequences.
 12. The method ofclaim 7, wherein said F1 genomic DNA is present separately in saidcollection.